The following is offered as background information only and is not admitted to be prior art to the present invention.
At present, many 2-dihydroindolinone derivatives have been attemped to identify as protein kinase inhibitors, which are widely used in the treatment of a variety of diseases associated with abnormal kinase activity, such as cancer, psoriasis, hepatocirrhosis, diabetes, angiogenesis, ophthalmological disease, rheumatoid arthritis and other inflammatory disorders, immune disease, cardiovascular disease, e.g. atherosclerosis, and a variety of kidney diseases. Of which, many indirubin derivatives (PCT WO2001037819, PCT WO2002092079), 3-methylenepyrrole-2-dihydroindolinone derivatives (U.S. Pat. No. 6,642,251, PCT WO2001060814, PCT WO2003035009, PCT WO2005053686), 3-pyrrolo[b]cyclopentylene-2-dihydroindolinone derivatives (PCT WO2005016875), and other 2-dihydroindolinone derivatives (PCT WO 2000012084) etc, all are described as the kinase inhibitors for treating cancer.
Mammalian cells have similar molecular mechanisms to regulate cell proliferation, differentiation and death in the entire cell cycle. Of these, protein phosphorylation is a major mechanism for transmembrane or intracellular signal transduction, with the function of cell cycle regulation, while phosphorylation is regulated by protein kinases (PKs) and protein phosphatases. Protein kinases are the largest known family of enzymes in humans, with a conserved catalytic domain and various regulation modes. Protein kinases are enzymes catalyzing the transfer of the terminal (γ) phosphoryl group of ATP to specific amino acid residues of substrate. According to the specificity of these amino acid residues, these kinases are divided into 4 types, of which the main two types are serine/threonine kinases (STKs) and protein tyrosine kinases (PTKs). In eukaryotes, there is physical segregation and distance between cell surface receptors and nuclear transcription. Extracellular signals affect the cascades of some protein kinases with multi-step phosphorylation, and finally alter the activity of transcription factors to activate or block gene transcription. Protein tyrosine kinases and protein serine/threonine kinases play an important role in the normal signal transduction process and their aberrant expression will result in a wide array of disorders and diseases such as cancer, arteriosclerosis, psoriasis, inflammatory responses and so on. Thus, it is a novel therapy strategy to regulate kinase activity and restore the physiological balance.
The family of protein tyrosine kinases, consisting of transmembrane receptors (receptor tyrosine kinases, RTKs) and cytoplasmic forms (non-receptor tyrosine kinases, CTKs), are involved in cellular signal transduction. The protein kinase complement of the human genome (kinome) consists of 30 tyrosine kinase families containing about 90 distinct protein tyrosine kinases (PTKs), of which 58 members are receptor tyrosine kinases. For a more complete discussion of tyrosine kinases, see Manning G, Science, 2002, 298:1912 which is incorporated by reference, including any drawings, as if fully set forth herein. Receptor tyrosine kinase is a class of transmembrane protein with cytoplasmic region and an extracellular portion which is composed of a very large protein domain binding to extracellular ligands e.g. a soluble or membrane-bound polypeptide, including insulin and a variety of growth factor. A Cytoplasmic portion contains the tyrosine kinase catalytic domain with autophosphorylation site, whose intrinsic catalytic activity that is activated upon ligand binding. Receptor tyrosine kinases include EGFR (epidermal growth factor receptor), VEGFR (vascular endothelial growth factor receptor), PDGFR (platelet-derived growth factor receptor), FGFR (fibroblast growth factor receptor) and so on. The most important downstream signaling cascades activated by RTKs include the Ras-extracellular ERK/MAPK pathway, the PI-3′ kinase-AKT and the JAK/STAT pathway. PTKs provide communication signals that link all these pathways ultimately leading to regulation of gene transcription. Additional cascades may also be utilized. Through a different regulatory mechanism, non-receptor tyrosine kinases (CTKs) participate in response to extracellular signals by physically associating with transmembrane receptors (Grosios k, et al, Drugs Fut, 2003, 28:679).
These phosphorylated tyrosine residues serve as docking sites for phosphotyrosine binding domains (e.g., Src homology 2 and 3 [SH2 and SH3] and phosphotyrosine binding [PT-3] domains) found in a number of intracellular signaling proteins (e.g., Shc, Grb2, Src, Cbl, phospholipase Cg and phoshoinositol-3′ [PI-3′ kinase]). Assembly of activated complexes at the membrane initiates several cascades which are the key to downstream signaling and biological response. Formation of homo- or heterodimers is also possible. Receptors lacking catalytic activity can be coupled to nonreceptor PTKs via noncovalent association with the cytoplasmic domain of a receptor subunit, thus forming “binary” receptors. The most important downstream signaling cascades activated by RTKs include the Ras-extracellular regulated kinase (ERK)-mitogen activated (MAP) kinase pathway, the PI-3′ kinase-AKT and the JAK/STAT pathway. PTKs provide communication signals that link all these pathways ultimately leading to regulation of gene transcription. Additional cascades may also be utilized. For example, the InsR utilizes the adenylyl cyclase signaling system which, in turn, activates cAMP-dependent serine-threonine specific protein kinases. (Grosios k, et al, Drugs Fut, 2003, 28:679)
Non-receptor tyrosine kinases (CTKs) participate in response to extracellular signals by physically associating with transmembrane receptors, such as hormone, cytokine and growth factor receptors. They are then activated when these receptors are bound by extracellular ligands or cell adhesion components at particular phases of the cell cycle.
In normal cells, activated RTKs are rapidly internalized away from the cell surface and are subject to modifications that inhibit their enzymatic activity. This ensures that activation of signal cascades are only transient and the cell returns to its non-stimulated state in a timely fashion. However, a variety of structural alterations ranging from single amino acid substitutions to large deletions, or deregulation of inhibitory signals and autocontrol mechanisms, can lock kinases into the activated form in which the kinase domain is always active. A number of diseases have been shown to be due to mutations that activate or lead to misexpression/overexpression of PTKs. During molecular characterization of malignancies, approximately half of all known PTKs such as EGF, ErbB2, Ret, Kit, Src, Abl, PDGFR, VEGF1/2/3, FGFR1/2/3, etc, have been found in either mutated or overexpressed forms including sporadic cases. Clinical studies also show over-expression or disorders of PTKs is of important reference value for the prognosis of cancer patients and symptoms prediction (Madhusudan S, et al, Clin Biochem, 2004, 37:618). In summary, tyrosine kinases are very important for physiological self-regulation and gene mutation/rearrangemen may lead to the disorder or over-expression of PTKs, then result in the occurrence of diseases, so the agonist or antagonist of PTKs can be used in the treatment.
Irrespective of the underlying genetic alteration, the outcome i.e., altered, aberrant or inappropriate receptor presence, gives rise to respective disease phenotypes (e.g., cancer). This is not however, maintained only by receptor deregulation but also in the context of the whole cell circuit and intra-/intercellular communications, i.e., a multitude of paracrine and autocrine communications. Growth factors (e.g., EGF, VEGF, PDGF) and their receptors are frequently overexpressed in cancers and their coexpression is often associated with tumor cell proliferation and other tumor parameters such as angiogenesis and metastasis.
Angiogenesis is a physiological process involving the growth of new blood vessels from pre-existing vessels. Normal angiogenesis only occurs in some particular short-term physiological processes, such as reproduction and wound healing etc. However aberrant angiogenesis is one of the pathological manifestations of some diseases including malignant tumor, rheumatoid arthritis, diabetic retinopathy and so on. Based on a large number of clinical practice and experiments, Folkman brought forth a hypothesis that tumor growth required angiogenesis: due to the lack of neovascularization, the diameter of tumors in the early stage of formation is limited in 2˜3 mm and cells number is less than one million; when entering the angiogenesis stage through the mediation of tumor angiogenesis factor (Tumor-angiogenesis Factor, TAF) secreted by tumor cells, the tumor can grow rapidly with adequate supply of oxygen and nutrients (Folkman J, N Engl J Med, 1971, 285:1182). Thus anti-angiogenesis therapy is a new anti-cancer strategy by blocking TAF.
Tumor cells can secrete a variety of angiogenic factors, which interact with each other. Vascular endothelial growth factor (VEGF) is thereinto the most specific angiogenic factors with highest activity and other angiogenic factors exert angiogenic effect mostly by enhancing the expression of VEGF (Zhang Q X, et al, J Surg Res, 1997, 67:147). VEGF is expressed in the vast majority of tumor cells and a variety of tissues such as lung, spleen, kidney, liver, etc. The expression of VEGF is regulated by many factors, of which hypoxia is by far the strongest induced effect. In addition, the growth factors following such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), keratinocyte growth factor (KGF), placental growth factor (PLGF), transforming growth factor β (TGFβ), insulin-like growth factor-1 (IGF-1), tumor necrosis factor α (TNFα), interleukin (IL)-1β, IL-6 and NO can also promote the expression of VEGF. But interferon-α (IFN-α), IL-10, IL-12, etc can inhibit the expression of VEGF. Furthermore, oncogene ras, raf, src, anti-oncogene vHL and p53 mutations can increase the expression of VEGF (Neufeld G, et al, FASEB J, 1999, 13:9).
VEGF receptor (VEGFR) are known to have three members: VEGFR-1/Flt-1, VEGFR-2 (Flk-1/KDR) and VEGFR-3/Flt-4, in which VEGFR-1 and VEGFR-2 are expressed specially in vascular endothelial cells and associated with angiogenesis closely. VEGFR, a member of receptor tyrosine kinase family, is a class of transmembrane protein with a cytoplasmic region, and its expression is induced by VEGF. Compared to a low level of expression in normal human tissues, both VEGF and its receptors (VEGFR) show the high expression level in the vast majority of malignant tumors. Furthermore, VEGFR is expressed not only in vascular endothelial cells, but also in tumor cells. So besides paracrine, it indicates the existence of autocrine pathway. As a vascular endothelial cell-specific mitogen, VEGF secreted by the malignant cells acts on VEGF receptor in vascular endothelial cells of adjacent stromal tissue, that promotes vascular endothelial cell division and proliferation, and induces tumor angiogenesis. Moreover, it also increases vascular permeability, promotes tumor growth and metastasis, then acts on tumor cells directly and stimulates tumor cell growth. (Rong L, Foreign Medical Sciences•Section of Pathophysiology and Clinical Medicine, 2002, 22:4475 and references cited)
Mutatioms in PKs and cross-talk in signal proteins are also involved in diseases other than cancer. Mutational inactivation of nonreceptor tyrosine kinase is observed in several immunodeficiencies. Inactivation of both copies of JAK3 causes severe combined immunodeficiency (Leonard W J, Nat Rev Immunol, 2001, 1:200; Leonard W J, Int J Hematol, 2001, 73:271). Mutation in the Bruton tyrosine kinase (BTK, also known as BPK or ATK), a member of the src family and a key regulator of B-cell maturation, causes X-linked Agammaglobulinemia (Cheng G, et al, Proc Natl Acad Sci USA, 1994, 91:8152; Maas A, et al, J Immunol, 1999, 162:6526). The physiological role of PTK in CNS signaling also suggests that deregulation of these proteins might also be involved in related disorders. This is supported by the observation that neuregulin-1 and ErbB4 immunoreactivity is associated with neuritic plaques in the Alzheimer's disease (Ferguson S S, Trends Neurosci, 2003, 26:119; Chaudhury A R, et al, J Neuropathol Exp Neurol, 2003, 62:42). Abnormal regulation of Insulin-like growth factor (IGFs) and its regulatory protein secreted by the cardiovascular system may lead to coronary atherosclerosis and the occurrence and development of restenosis. The role of IGFs is mediated by specific membrane receptors, in which IGF receptor-I shows tyrosine kinase activity and appears in smooth muscle cells, inflammatory cells and arterial endothelial cells in atherosclerotic injury (Bayes-genis A, et al, Circ Res, 2000, 86:125; Bayes-genis A, et al, Artherio Thromb and Vascu Biol, 2001, 21:335; Che W Y, et al, Circ Res, 2002, 90:1222). Vascular endothelial growth factor and its receptors expressed in a variety of rheumatoid arthritis cells are a key factor in the pathological angiogenesis of rheumatoid arthritis (De Bandt M, et al, J Immunol, 2003, 1712:4853). Jak2 is a cytoplasmic, non-receptor tyrosine kinase and its mutation causes at least three diseases, such as polycythemia vera (PV), idiopathic myelofibrosis (IMF), essential thrombocythemia (ET) as well as some other atypical myeloproliferative disorders (MPD). Mutation in the tyrosine kinase domain of fibroblast growth factor receptor would lead to the most common hereditary dwarfism—bone Dyschondroplasia (Shiang R, et al, Cell, 1994, 78: 335).
On the other hand, a number of diseases are due to insufficient PTK signaling, such as non-insulin-dependent diabetes and peripheral neuropathies, and in such cases methods to enhance signaling could serve as viable therapies (Hunter T, Cell, 2000, 100:111). This is also a very attractive possibility for other angiogenesis-related conditions, including certain cardiovascular diseases where stimulation of angiogenesis might be required rather than inhibition.
With the in-depth study on molecular biology, it is an effective way to inhibit tumor cell proliferation and treat cancer by regulating the cellular signal transduction, mediating the function of growth factor and regulating oncogene expression at the molecular level. It is effective to weaken the effect of abnormal signal pathway, inhibit tumor growth and promote tumor cell death. Up to now, more than half of proto-oncogenes encode tyrosine kinase proteins. They participate in cellular signal transduction by phosphorylation and dephosphorylation, while in the process of tumorigenesis, mutation or over-expression of PTK can transform normal cells into cancer cells and promote growth and mitosis of tumor cells.
At the same time, the growth and metastasis of malignant tumors rely on the adequate nutrient supply through the new peripheral blood vessels. The process of tumor angiogenesis can generally be divided into two stages: preangiogenesis period and angiogenesis period. Transformation of these two stages is called as “angiogenic switch”. It is a key role in the process of deterioration that tumor cells switch to the angiogenic phenotype. Tumor cells can not get enough nutrients and discharge metabolites without peripheral angiogenesis, and mainly survive on oxygen and nutrients dispersed around cells, thus tumors can not grow beyond 1-2 mm in diameter. Once switch to angiogenic phenotype, tumors without blood vessels can grow rapidly utilizing the nutrients from the blood. Furthermore, these malignant cells can induce phenotypic changes of other cells, such as endothelial cells, then promote the formation of new blood vessels. Angiogenic factors participate in the regulation of angiogenic switch, cause endothelial cell migration, proliferation and morphological change, then initiate the generation of tumor blood vessels. All the known angiogenic factors mainly are the ligands of PTKs, such as VEGF, bFGF, PD-ECGF, etc (Bergers G, et al, Nat Rev Cancer, 2003, 3:401). Therefor it is an effective therapy to prevent the formation of tumor angiogenesis and control the growth of malignant tumors by using the tyrosine kinase inhibitors as anti-angiogenesis substances.
TKs play an important role in carcinogenic transformation of cells and relate to the occurrence and progress of tumors directly or indirectly, so it is especially appropriate for TKs inhibitors used in the treatment of tumors.
The serine/threonine kinases (STKs) are kinases family members that catalyze the phosphorylation of specific serine and threonine residues. STKs, like the non-receptor PTKs, are predominantly intracellular although there are a few receptor kinases of the STK type. STKs are the most common of the cytosolic kinases, i.e., kinases that perform their function in that part of the cytoplasm other than the cytoplasmic organelles and cytoskeleton. STKs affect the internal biochemistry of the cell, often as a down-line response to a PTK event. STKs have been implicated in the signaling process which initiates DNA synthesis and subsequent mitosis leading to cell proliferation. Additionally STKs are associated with many types of cancers, such as breast cancer etc. (Cance et al, Int. J. Cancer, 1993, 55, 571).
PTKs and STKs have all been implicated in a host of pathogenic conditions including, significantly, cancer. Other pathogenic conditions which have been associated with PKs include, without limitation, psoriasis, hepatocirrhosis, diabetes, angiogenesis, restenosis, ophthalmological disease, rheumatoid arthritis and other inflammatory disorders, immune disease, cardiovascular disease, e.g. atherosclerosis, and a variety of kidney diseases.
Presently, many 2-dihydroindolinone derivatives attemped to identify as protein kinase inhibitors, such as indirubin derivatives (PCT WO2001037819, PCT WO2002092079), 3-methylenepyrrole-2-dihydroindolinone derivatives (U.S. Pat. No. 6,642,251, PCT WO2001060814, PCT WO2003035009, PCT WO2005053686), 3-pyrrolo[b]cyclopentylene-2-dihydroindolinone derivatives (PCT WO2005016875), and other 2-dihydroindolinone derivatives (PCT WO 2000012084) etc, all are described as STK or PTK inhibitors for treating cancer.